One approach to improve the pneumococcal vaccine is to supplement the polysaccharide vaccine with protein antigens. Pneumolysin is an important virulence factor produced by most pneumococci. Thus, inactivated pneumolysin can act as a carrier protein to link covalently with the pneumococcal PS to form a PS-protein conjugate. The inactivated pneumolysin was prepared from the mutant ply gene cloned and expressed in E. coli (pneumolysoid, pdB), or treatment of 19F pneumolysin with 0.1% formaldehyde at 37 C for 30-60 min. The protein was covalently linked to PS after derivatization of the PS with a thiol group and the protein with a bromoacetyl group. The conjugates, in which 9V PS or 19F PS was linked to the inactivated pneumolysin were injected into mice during pregnancy and/or lactation, and into their offspring during early life. The anti-PS and anti-pneumolysin titers of the immunized young mice were significantly higher than those of non-immunized controls. When the young mice were challenged with type 9V or type 19F pneumococci the bacteria were cleared more effectively from the blood of immunized mice than from control mice. The survival rate for the immunized mice challenging with 19F cells was also longer than that for the control group. These results indicated that the protective immunity could be induced in young mice by immunization with the conjugate during gestation, lactation or early infancy. To study the effect of molecular size on the antibody response, type 19F PS was cleaved into different sizes from PS to oligosaccharide, then conjugated with inactivated 19F pneumolysin. The 19F IgG antibody response was determined. The conjugate that used large molecular size PS produced higher serum levels of total 19F IgG as well as 3.5 to 4.5 fold higher IgG1 and 2.5 to 16.8 fold higher IgG3 antibodies in young mice, than the conjugates using smaller PS or oligosaccharide.